The ends of mammalian chromosomes terminate in long arrays of TTAGGG repeats that have associated with them specific DNA binding proteins. These nucleoprotein complexes are known as telomeres and they function to preserve the integrity of chromosomes during the cell cycle by allowing the proper segregation during cell division. More specifically, telomeres shield the chromosome ends from degradation or end-on fusion, prevent the activation of DNA damage checkpoints, and modulate activity of telomerase, an enzyme that maintains the length of telomeres. Without this enzymatic activity, telomeres shorten at each cell division because the replication machinery fails to replicate DNA ends.
Human somatic cell chromosomes undergo normal telomere shortening at each cell division, and it is believed that this process is a tumor suppression mechanism that limits the number of potential replication cycles of the cell. However, in some cancers it has been demonstrated that telomeres do not undergo replication-associated shortening resulting in the transformed phenotype and the subsequent development of tumorigenesis. On the other hand, the complete loss of telomeric DNA in tumors has also been observed, collectively suggesting that any modification to telomere length homeostasis can contribute to the carcinogenic phenotype.
The activity of the enzyme, telomerase, is the best understood mechanism to maintain telomere length. Telomerase is an RNA-dependent DNA polymerase that, in vertebrates, elongates the 3' end of preexisting telomeres using an intrinsic RNA molecule as a template. This enzyme is a ribonucleoprotein complex, the protein component of which contains the catalytic domain of the complex (reviewed in Nugent and Lundblad, Genes Dev. 1998, 12(8), 1073-85; Bryan and Cech, Curr. Opin. Cell Biol., 1999, 11 (3), 318-24). The expression of telomerase is repressed in normal human somatic cells but is reactivated during tumor progression. For this reason, much effort is currently focused on the characterization of the telomerase complex, especially the catalytic subunit.
The catalytic subunit of telomerase, TERT or Telomerase Reverse Transcriptase was isolated and characterized simultaneously by several investigators and is therefore identified in the art by several names including TRT/TERT (Nakamura, Morin, et al., Science, 1997, 277(5328), 955-9), EST2 (Meyerson, Counter, et al., Cell, 1997, 90(4), 785-95), TCS1 (Kilian, Bowtell, et al., Hum. Mol. Genet., 1997, 6(12), 2011-9) and TP2 (Harrington, Zhou, et al., Genes Dev., 1997, 11(23), 3109-15). The TERT nucleotide and polypeptide sequence is further disclosed in the PCT Publications, WO 98/21343 and WO 98/37181 and in the UK Patent Application GB 2317891 A to (Cech, Lingner et al. 1998; Counter, Meyerson et al. 1998 and Harrington and Robinson, 1998).
Further disclosed in the PCT Publication, WO 98/21343 are a nucleic acid molecule that hybridizes to the nucleic acid sequence of TERT, vectors encoding TERT, host cells capable of expression said vectors, methods of increasing the proliferation of a cell comprising expressing a nucleic acid encoding TERT, methods of increasing and methods of decreasing telomerase activity in a cell comprising expressing a biologically active fragment of TERT or a mutant of TERT, respectively and nucleic acid molecules encoding mutant TERT polypeptides (Harrington and Robinson, 1998).
In the PCT Publication, WO 98/37181 are disclosed the DNA encoding the catalytic subunit of eukaryotic or yeast telomerase, polynucleotides and polypepeptides encoding TERT, isolated DNA which hybridizes to the complement of TERT under high stringency, isolated DNA and mRNA which encodes the human TERT gene, nucleic acid probes to the TERT gene, methods for assessing cells for malignancy comprising measuring the amount of TERT expression in said cells and methods of reducing expression of TERT protien by administering drugs which inhibit or bind TERT RNA and prevent or reduce the production of the TERT protein said drugs including antisense molecules. Also disclosed are methods of using said drugs to treat cancer in an individual and methods of increasing or decreasing the lifespan of a cell administering said drugs to a cell (Counter, Meyerson et al., 1998).
The polynucleotide and polypeptide encoding the TERT gene and protein, respectively, are also disclosed in the UK Patent Application GB 2317891 A. Also disclosed are vectors encoding TERT, cells expressing said vectors, antibodies to TERT protein, methods of detecting TERT expression, methods of increasing the proliferation of cells by using an agent which increases the expression of TERT, the use of an inhibitor of telomerase in the treatment of a condition associated with an elevated level of telomerase activity and the use of proteins or fragments thereof of TERT in the manufacture of a medicament for inhibiting ageing or cancer (Cech, Lingner et al., 1998).
TERT expression is regulated by both the Sp1 and c-myc transcription factors, genes which are frequently deregulated in human tumors (Wang, Xie, et al., Genes Dev., 1998, 12(12), 1769-74; Wu, Grandori, et al., Nat. Genet., 1999, 21(2), 220-4; Kyo, Takakura, et al., Nucleic Acids Res., 2000, 28(3), 669-677) and reviewed in (Cerni, Mutat. Res., 2000, 462(1), 31-47). These factors bind at the promoter region of TERT which has also been characterized (Devereux, Horikawa, et al., Cancer Res., 1999, 59(24), 6087-90; Horikawa, Cable, et al., Cancer Res., 1999, 59(4), 826-30; Wick, Zubov, et al., Gene, 1999, 232(1), 97-106). Disclosed in the UK Patent Application GB 2321642 A are the polynucleotide encoding the TERT promoter sequence, polynucleotides wherein the TERT promoter is linked to a gene encoding a protein that renders cells sensitive to a nontoxic drug, polynucleotides wherein the TERT promoter is linked to a gene encoding a protein that is detectable by fluorescence, phosphorescence or by possessing an enzyme activity. Also generally disclosed are polynucleotides from about 15 nucleotides in length to about 100 nucleotides in length complementary to the TERT promoter sequence, as well as antisense oligonucleotides, and methods of killing a cell using said antisense oligonucleotides (Cech, Lingner et al., 1998).
The TERT mRNA transcript undergoes alternative splicing in different cell types and this is believed to be one mode of regulation of TERT expression (Ulaner, Hu, et al., Cancer Res., 1998, 58(18), 4168-72; Brenner, Wolny, et al., Mol. Hum. Reprod., 1999, 5(9), 845-50; Ulaner, Hu, et al., Int. J. Cancer, 2000, 85(3), 330-5).
TERT has also been shown to be a natural substrate for Akt kinase, a serine/threonine kinase first identified as an oncogene because of its ability to induce transformation in normal cells (Kang, Kwon, et al., J. Biol. Chem., 1999, 274(19), 13085-90).
Increased levels of TERT have been associated with many disorders and are considered to be a reliable marker for several types of cancer including urinary bladder cancer (Suzuki, Suzuki, et al., J. Urol., 1999, 162 (6), 2217-20), renal cell carcinoma (Kanaya, Kyo, et al., Int. J. Cancer, 1998, 78(5), 539-43), malignant lymphoma (Harada, Kurisu, et al., Cancer 1999, 86(6), 1050-5), acute myelogenous leukemia (Xu, Gruber, et al., Br. J. Haematol., 1998, 102(5), 1367-75), breast cancer (Umbricht, Sherman, et al., Oncogene, 1999, 18(22), 3407-14), head and neck squamous cell carcinomas (Thurnher, Knerer, et al., Acta Otolaryngol., 1998, 118(3), 423-7), neuroblastomas (Poremba, Willenbring, et al., Ann. Oncol., 1999, 10 (6), 715-21), hepatocellular carcinoma (Hisatomi, Nagao, et al., Int. J. Oncol., 1999, 14(4), 727-32), colorectal cancer (Tahara, Yasui, et al., Oncogene, 1999, 18 (8), 1561-7), gastric cancer (Jong, Park, et al., Cancer, 1999, 86(4), 559-65), thyroid neoplasms (Saji, Xydas, et al., Clin. Cancer Res., 1999, 5(6), 1483-9), cervical and ovarian carcinomas (Snijders, van Duin, et al., Cancer Res. , 1998, 58(17), 3812-8; Takakura, Kyo, et al., Cancer Res., 1998, 58(7), 1558-61; Kyo, Kanaya, et al., Int. J. Cancer, 1999, 80(6), 804-9; Park, Riethdorf, et al., Int. J. Cancer, 1999, 84(4), 426-31) and skin tumors (Wu, Ichihashi, et al., Cancer, 1999, 86(10), 2038-44).
Currently, strategies aimed at modulating TERT function have involved the use of antibodies, dominant negative mutants of the TERT protein, gene knockouts in mice and antisense molecules.
Inducible dominant negative mutants of TERT have been shown to radically reduce the telomerase activity, reduce telomere length and increase death of tumor cells (Hahn, Stewart, et al., Nat. Med. 1999, 5(10), 1164-70; Zhang, Mar, et al., Genes Dev., 1999, 13(18), 2388-99).
Mice lacking the TERT gene showed phenotypes that were apparently normal during the early generations and all tissues examined from these mice lacked telomerase activity. These studies indicated that TERT is the only gene encoding the catalytic function of the telomerase complex (Yuan, Nikaido, et al., Genes Cells, 1999, 4(10), 563-72).
Disclosed in the PCT publication WO 99/50279 are a series of antisense phosphorothioate oligonucleotides, 30 nucleotides in length, targeting the nucleic acid encoding TERT wherein the polynucleotide inhibits telomerase activity or expression by at least 50% (Cech, Lingner et al., 1999).
Other less specific inhibitors include reverse transcriptase inhibitors (RTI) (Beltz, Moran et al., Anticancer Res., 1999, 19(4B), 3205-11; Murakami, Nagai, et al., Eur. J. Cancer, 1999, 35(6), 1027-34; Yegorov, Akimov, et al., Anticancer Drug Des., 1999, 14(4), 305-16), isothiazolone derivatives (Hayakawa, Nozawa, et al., Biochemistry 1999, 38(35), 11501-7), heparin (Engelberg, Cancer, 1999, 85(2), 257-72), gonadotropin releasing hormone (Ohta, Sakamoto, et al., Cancer Lett., 1998, 134(1), 111-8) and deoxynucleoside analogs (Pai, Pai, et al., Cancer Res., 1998, 58(9), 1909-13).
There remains, however, a long felt need for additional agents capable of effectively inhibiting TERT function and antisense technology is emerging as an effective means for reducing the expression of specific gene products. This technology may prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of TERT expression.
The present invention provides compositions and methods for modulating TERT expression, including modulation of the alternatively spliced form of TERT.